Progressive Multifocal Leukoencephalopathy (PML), caused by the reactivation of JC virus (JCV), remains a life-threatening AIDS-defining infection of the central nervous system. In addition, PML also occurs in HIV- negative patients treated with monoclonal antibodies for cancer or autoimmune diseases. Although the shedding of JCV in the urine of healthy individual is asymptomatic, the reactivation of JCV in the brain of PML patients causes severe damages from destruction of myelin. Therefore, halting JC viral replication in the brain can prevent progression to PML. However, knowledge of the virologic and immunologic events occurring in the CNS prior to onset of PML is limited; better understanding of PML pathogenesis is hampered by the lack of an animal model. Our overall goal is to generate a mouse model of PML. Our central hypothesis is that a novel chimeric murine-JC polyomavirus, harboring the murine replication machinery and the neuropathogenic type JCV capsid will cause productive and lytic infection of mouse oligodendrocytes, and recapitulate the pathogenesis of PML in mice. The rationale is based on previous studies of murine polyomavirus and JCV strains isolated from the CSF of PML patients, indicating altered pathogenicity due to changes in the VP1 capsid, as well as our preliminary data demonstrating the creation of a novel chimeric murine-JC polyomavirus, rMPyV-JCVMad. A mouse model of PML is urgently needed to further our understanding of immune events leading to JCV reactivation and the development of PML. Such small animal model will be crucial in devising preventive and therapeutic strategies for PML in HIV+ and other immunosuppressed patients. Based on our strong preliminary data, we will test this hypothesis by pursuing these specific aims: Aim 1. Characterize rMPyV-JCVMad chimeric virus in vitro. Aim 2. Develop a mouse model of PML. The proposed studies draw upon our extensive experiences in molecular virology, histology, and prior mouse studies. The development of new tools, including the novel chimeric murine-JC polyomavirus will also be useful to other researchers and will pave the way for the discovery of new targets for prevention and treatment of PML. This proposed work will produce the first animal model of PML, which will expand our understanding of JCV pathogenesis in the CNS, and reveal new targets for therapeutic interventions.